- Email: [email protected]
Paradoxical Effects of Platelets Aggregation on Thiol Redox Status
this study, we show that nitrite (10-25μM) increases cellular cAMP levels in cardiomyocytes leading to PKA activation. This cAMP increase is due to the inhibition of phosphodiesterase activity. Interestingly, the mitochondrially localized phosphodiesterase appears to be a major target for nitrite. Further, nitrite increases the expression of A-kinase anchoring protein (AKAP121), which localizes PKA to the outer mitochondrial membrane. Consistent with the mitochondrial targeting of PKA, we show that nitrite induces the phosphorylation of Ser58 on mitochondrial complex IV (a known PKA target), leading to augmented basal and maximal respiration. Ongoing studies are investigating the mechanism by which nitrite increases AKAP121 expression as well as which PDE isoform is inhibited by nitrite. These data demonstrate that nitrite can be a versatile signaling molecule, not only by inducing protein nitration and nitrosylation but also through modulating protein expression and phosphorylation. Further, these data contribute to the understanding of the mechanism by which nitrite selectively regulates mitochondrial function and metabolism to mediate protective cardiovascular signaling. doi: 10.1016/j.freeradbiomed.2014.10.421
107 Expression and Redox Contribution of Cytoglobin to the Smooth Muscle Fibroproliferative Phenotype during Vascular Remodeling in Human Blood Vessels Timothy Reilly1, Frances L. Jourd'heuil1, Julia Steppich1, Diane Singer1, Xiaochun Long1, Paul Kreienberg1,2, David Conti1, Roman Ginnan1, Harold Singer 1, Arif Asif1, and David Jourd'heuil1 1 Albany Medical College, USA, 2Albany Vascular Group, USA Background: Increase nitroxidative stress contributes to the smooth muscle fibroproliferative and inflammatory phenotype during pathological vascular remodeling. Cytoglobin (CYGB) is a hemoglobin paralog, which has been shown to preserve cell viability and proliferation under conditions of nitroxidative stress. The goal of the present study was (1) to determine whether CYGB is expressed in healthy human vessels (2) to examine changes in CYGB expression in humans during vasculopathic states associated with neointimal hyperplasia in the settings of dialysis access and (3) to evaluate CYGB cytoprotective functions in the context of increase reactive oxygen species (ROS) and nitric oxide (NO) production in human smooth muscle. Results: Cytoglobin mRNA and protein were detected in human veins and arteries. In veins obtained from arteriovenous fistulae (AVFs) from hemodialysis patients, CYGB protein levels were increased in veins from failed hyperplastic AVFs compared to veins obtained at creation with significant association with both medial smooth muscle and neointimal cells. Among several cytokines, interferon-gamma was the only stimuli which consistently increased CYGB protein expression in subcultured human venous and arterial smooth muscle cells. Using molecular strategies to decrease CYGB protein levels, we found no evidence that CYGB regulated smooth muscle cell proliferation in response to serum, even in the presence of interferon gamma. However, siRNA depletion of CYGB increases smooth muscle cell death in response to nitroxidative stress. Conclusions: Our study demonstrates for the first time that the stress-responsive protein CYGB is expressed in both arterial and venous beds in humans. CYGB expression might be increased in human vessels during conditions associated with pathological vascular remodeling. Collectively, our findings suggest that smooth muscle CYGB regulates cell viability during proinflammatory conditions associated with increased ROS and NO production. Funding: American Heart Association and DCI Paul Teschan
Research Fund.
doi: 10.1016/j.freeradbiomed.2014.10.422
108 Paradoxical Effects of Platelets Aggregation on Thiol Redox Status Hirotaka Sawada1,2, Saranya Ravi1,2, Michelle S Johnson1,2, and Victor M Darley-Usmar1,2 1 Department of Pathology, University of Alabama at Birmingham, USA, 2Center for Free Radical Biology, University of Alabama at Birmingham, USA Platelets are designed to rapidly transform from a quiescent phase to a strongly activated condition during the process of thrombosis. This thrombotic event involves the activation of numerous enzymes which are pro-oxidative; such as nitric oxide synthase and cyclooxygenase. Most cells under this condition would exhibit the markers of severe oxidative stress such as protein thiol oxidation. However, the platelet is functioning biologically under pro-oxidative conditions and we hypothesized that it can engage antioxidant protective pathways during aggregation. To test this, we evaluated the effect of thrombin dependent aggregation on redox status using metabolomics, measurement of thiol oxidation state, and mitochondrial bioenergetics. We isolated human platelets and incubated them with thrombin (0.5 U/mL) to achieve maximum aggregation. This resulted in a profound increase in the amount of glucose-6phosphate consistent with activation of the pentose phosphate pathway necessary for the maintenance of NADPH. In addition, we found that glutaminolysis was increased with 1.5-fold increase in glutamine and 3.2-fold increase in glutamate. This finding led to the hypothesis that the redox status is maintained in activated platelets since glutamate is a precursor of glutathione, and the glycolysis can provide reducing equivalents from NADPH via the pentose phosphate pathway. Furthermore, we activated the platelets with thrombin and measured the total thiols which increased dramatically (2- to 3-fold) depending upon the donor along with an increase in glutathione levels. Taken together, these data suggest that there is an adaptive response to the oxidative stress associated with platelet aggregation involving the activation of glycolysis and glutaminolysis. doi: 10.1016/j.freeradbiomed.2014.10.423
109 Circulating Oxidized Albumin - A Biomarker of Uremia and a Mediator of Endothelial Injury Faiga Magzal 1, Regina Michelis 2, Bayta Kristal1,2, Snait Tamir 3, Andrea Szuchman-Sapir 3, and Shifra Sela1,2 1 Faculty of Medicine, Bar Ilan University, Safed, Israel, 2Galilee Medical Center, Israel, 3Laboratory of Human Health and Nutrition Sciences, MIGAL - Galilee Research Institute, Kiryat Shemona, Israel Oxidative stress (OS) plays a critical role in the progression of chronic kidney disease (CKD). We have shown that human serum albumin (HSA), is modified by OS in these patients, resulting in altered conformation and decreased activity. Our hypothesis is that the nature of the post-translational modifications of circulating HSA correlate with the severity of kidney disease. This modified HSA plays a role in endothelial dysfunction underlying cardiovascular diseases prevalent in this patient population. The aims were to characterize and identify the
SFRBM 2014
S53
107 Expression and Redox Contribution of Cytoglobin to the Smooth Muscle Fibroproliferative Phenotype during Vascular Remodeling in Human Blood Vessels Timothy Reilly1, Frances L. Jourd'heuil1, Julia Steppich1, Diane Singer1, Xiaochun Long1, Paul Kreienberg1,2, David Conti1, Roman Ginnan1, Harold Singer 1, Arif Asif1, and David Jourd'heuil1 1 Albany Medical College, USA, 2Albany Vascular Group, USA Background: Increase nitroxidative stress contributes to the smooth muscle fibroproliferative and inflammatory phenotype during pathological vascular remodeling. Cytoglobin (CYGB) is a hemoglobin paralog, which has been shown to preserve cell viability and proliferation under conditions of nitroxidative stress. The goal of the present study was (1) to determine whether CYGB is expressed in healthy human vessels (2) to examine changes in CYGB expression in humans during vasculopathic states associated with neointimal hyperplasia in the settings of dialysis access and (3) to evaluate CYGB cytoprotective functions in the context of increase reactive oxygen species (ROS) and nitric oxide (NO) production in human smooth muscle. Results: Cytoglobin mRNA and protein were detected in human veins and arteries. In veins obtained from arteriovenous fistulae (AVFs) from hemodialysis patients, CYGB protein levels were increased in veins from failed hyperplastic AVFs compared to veins obtained at creation with significant association with both medial smooth muscle and neointimal cells. Among several cytokines, interferon-gamma was the only stimuli which consistently increased CYGB protein expression in subcultured human venous and arterial smooth muscle cells. Using molecular strategies to decrease CYGB protein levels, we found no evidence that CYGB regulated smooth muscle cell proliferation in response to serum, even in the presence of interferon gamma. However, siRNA depletion of CYGB increases smooth muscle cell death in response to nitroxidative stress. Conclusions: Our study demonstrates for the first time that the stress-responsive protein CYGB is expressed in both arterial and venous beds in humans. CYGB expression might be increased in human vessels during conditions associated with pathological vascular remodeling. Collectively, our findings suggest that smooth muscle CYGB regulates cell viability during proinflammatory conditions associated with increased ROS and NO production. Funding: American Heart Association and DCI Paul Teschan
Research Fund.
doi: 10.1016/j.freeradbiomed.2014.10.422
108 Paradoxical Effects of Platelets Aggregation on Thiol Redox Status Hirotaka Sawada1,2, Saranya Ravi1,2, Michelle S Johnson1,2, and Victor M Darley-Usmar1,2 1 Department of Pathology, University of Alabama at Birmingham, USA, 2Center for Free Radical Biology, University of Alabama at Birmingham, USA Platelets are designed to rapidly transform from a quiescent phase to a strongly activated condition during the process of thrombosis. This thrombotic event involves the activation of numerous enzymes which are pro-oxidative; such as nitric oxide synthase and cyclooxygenase. Most cells under this condition would exhibit the markers of severe oxidative stress such as protein thiol oxidation. However, the platelet is functioning biologically under pro-oxidative conditions and we hypothesized that it can engage antioxidant protective pathways during aggregation. To test this, we evaluated the effect of thrombin dependent aggregation on redox status using metabolomics, measurement of thiol oxidation state, and mitochondrial bioenergetics. We isolated human platelets and incubated them with thrombin (0.5 U/mL) to achieve maximum aggregation. This resulted in a profound increase in the amount of glucose-6phosphate consistent with activation of the pentose phosphate pathway necessary for the maintenance of NADPH. In addition, we found that glutaminolysis was increased with 1.5-fold increase in glutamine and 3.2-fold increase in glutamate. This finding led to the hypothesis that the redox status is maintained in activated platelets since glutamate is a precursor of glutathione, and the glycolysis can provide reducing equivalents from NADPH via the pentose phosphate pathway. Furthermore, we activated the platelets with thrombin and measured the total thiols which increased dramatically (2- to 3-fold) depending upon the donor along with an increase in glutathione levels. Taken together, these data suggest that there is an adaptive response to the oxidative stress associated with platelet aggregation involving the activation of glycolysis and glutaminolysis. doi: 10.1016/j.freeradbiomed.2014.10.423
109 Circulating Oxidized Albumin - A Biomarker of Uremia and a Mediator of Endothelial Injury Faiga Magzal 1, Regina Michelis 2, Bayta Kristal1,2, Snait Tamir 3, Andrea Szuchman-Sapir 3, and Shifra Sela1,2 1 Faculty of Medicine, Bar Ilan University, Safed, Israel, 2Galilee Medical Center, Israel, 3Laboratory of Human Health and Nutrition Sciences, MIGAL - Galilee Research Institute, Kiryat Shemona, Israel Oxidative stress (OS) plays a critical role in the progression of chronic kidney disease (CKD). We have shown that human serum albumin (HSA), is modified by OS in these patients, resulting in altered conformation and decreased activity. Our hypothesis is that the nature of the post-translational modifications of circulating HSA correlate with the severity of kidney disease. This modified HSA plays a role in endothelial dysfunction underlying cardiovascular diseases prevalent in this patient population. The aims were to characterize and identify the
SFRBM 2014
S53